The activity of gamma-glutamyltransferase (GGT) is frequently upregulated in tumor cells after oxidative stress and may thus increase the availability of amino acids needed for biosynthesis of the antioxidant glutathione. As gamma-radiation of tumor cells can result in oxidative stress, we investigated whether such treatments modulate the enzyme level in colon carcinoma CC531 cells. Radiation of these cells blocked cell proliferation, increased cellular size, initiated apoptosis and upregulated GGT activity and protein levels in a dose- and time-related manner. A slight but significant increase in the cellular level of reactive oxygen species (ROS) was found directly after radiation but appeared not to cause the GGT elevation. Thus, other mechanisms than cellular oxidative stress appear to be responsible for the radiation-induced upregulation of GGT. Stable transfection of activated Ras in a human colon carcinoma cell line expressing wild-type Ras resulted in an increased GGT level, while a reduced enzyme level was demonstrated in another cell line with constitutively activated Ras after stably transfection with a dominant-negative Ras mutant. Moreover, addition of specific protein kinase inhibitors that blocked downstream targets PI-3K and MEK1/2 of Ras, prior to and after radiation, attenuated the radiation-induced activation of GGT. These results support a role for Ras, being frequently activated after radiation, in regulating the level of GGT and also indicate that GGT participates in radioresistance.